Exploring functional and structural features of chemically related natural prenylated hydroquinone and benzoic acid from Piper crassinervium (Piperaceae) on bacterial peroxiredoxin inhibition

Multiple drug resistance (MDR) bacterial strains are responsible by 1.2 million of human deaths all over the world. The pathogens possess efficient enzymes which are able to mitigate the toxicity of reactive oxygen species (ROS) produced by some antibiotics and the host immune cells. Among them, the bacterial peroxiredoxin alkyl hydroperoxide reductase C (AhpC) is able to decompose efficiently several kinds of hydroperoxides. To decompose their substrates AhpC use a reactive cysteine residue (peroxidatic cysteine—CysP) that together with two other polar residues (Thr/Ser and Arg) comprise the catalytic triad of these enzymes and are involved in the substrate targeting/stabilization to allow a bimolecular nucleophilic substitution (SN2) reaction. Additionally to the high efficiency the AhpC is very abundant in the cells and present virulent properties in some bacterial species. Despite the importance of AhpC in bacteria, few studies aimed at using natural compounds as inhibitors of this class of enzymes. Some natural products were identified as human isoforms, presenting as common characteristics a bulk hydrophobic moiety and an α, β-unsaturated carbonylic system able to perform a thiol-Michael reaction. In this work, we evaluated two chemically related natural products: 1,4-dihydroxy-2-(3’,7’-dimethyl-1’-oxo-2’E,6’-octadienyl) benzene (C1) and 4-hydroxy-2-(3’,7’-dimethyl-1’-oxo-2’E,6’-octadienyl) benzoic acid (C2), both were isolated from branches Piper crassinervium (Piperaceae), over the peroxidase activity of AhpC from Pseudomonas aeruginosa (PaAhpC) and Staphylococcus epidermidis (SeAhpC). By biochemical assays we show that although both compounds can perform the Michael addition reaction, only compound C2 was able to inhibit the PaAhpC peroxidase activity but not SeAhpC, presenting IC50 = 20.3 μM. SDS-PAGE analysis revealed that the compound was not able to perform a thiol-Michael addition, suggesting another inhibition behavior. Using computer-assisted simulations, we also show that an acidic group present in the structure of compound C2 may be involved in the stabilization by polar interactions with the Thr and Arg residues from the catalytic triad and several apolar interactions with hydrophobic residues. Finally, C2 was not able to interfere in the peroxidase activity of the isoform Prx2 from humans or even the thiol proteins of the Trx reducing system from Escherichia coli (EcTrx and EcTrxR), indicating specificity for P. aeruginosa AhpC.

Introduction such as antibodies and natural or synthetic low molecular weight compounds, have already been characterized as eukaryotic counterparts [27][28][29][30][31][32][33][34]. A common feature shared among low molecular weight compounds is a very bulky carbon skeleton, which may resemble natural oxidant substrates as organic hydroperoxides. Among the natural compounds identified to eukaryotic isoforms, three of them are ent-kauranes diterpenes (adenanthin, JM-202, and parvifoline AA). The inhibitory action of these compounds is based on the alkylation of the catalytic cysteines through a thiol-Michael addition involving an α,β-unsaturated carbonylic system from the inhibitors [29,30,33].
As part of our continuous studies concerning the identification of natural inhibitors of peroxiredoxins, we here isolated two chemically related natural compounds-1,4-dihydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzene (C1) and 4-hydroxy-2-(3',7'-dimethyl-1'oxo-2'E,6'-octadienyl) benzoic acid (C2) from branches of Piper crassinervium (Piperaceae). These chemicals were tested over the AhpCs containing Thr or Ser as part of the catalytic triad, using recombinant enzymes from Pseudomonas aeruginosa and Staphylococcus epidermidis, opportunistics Gram -negative and -positive bacteria involved in hospital acquired infections. Using biochemical approaches, we identified compound C2 as a novel natural product with inhibitory properties over PaAhpC but not over SeAhpC, that presents IC 50 in the order of 20.3 μM. We also demonstrated that the inhibitory properties were not due to the thiol-Michael addition mechanism over the catalytic cysteines, since the intramolecular disulfide The thiolate nucleophilicity of peroxidatic cysteine (Cys P ) is increased by hydrogen bonds with the catalytic triad Arg and Thr residues (i). The hydroperoxide (Hpx) is trapped by the Arg hydrogen bond which is able to target the Hpx to the Prx active site (ii). The shift of the Arg and Thr hydrogen bonds from the Cys P thiolate to the substrate stabilizes the Hpx and increase the thiolate reactivity enabling the S N 2 mechanism (iii). After the catalytic reaction, the Cys P is oxidized to cysteine sulfenic acid (Cys P -SOH) and the release of the leaving group (R-OH, meaning a water molecule in the case of hydrogen peroxide, or the alcohol derivative, in the case of organic hydroperoxides) is assisted by polar interaction with Arg (iv). The cysteine sulfenic acid formation triggers structural changes which allow the condensation among Cys P -SOH with Cys R -SH (v) resulting in the disulfide formation and concomitant release of a water molecule (vi). The asterisk ( � ) in Cys R , denotes the adjacent subunit of the homodimer. formation of the enzyme was preserved. We also evaluated the inhibitory effect of the compound C2 over the human isoform Prx2 and the thiol proteins Trx and TrxR from bacteria, and no significant inhibition was detected in the tested conditions, suggesting specificity to PaAhpC. Finally, we executed computer assisted molecular simulations to investigate a possible mode of binding, which revealed that compound C2 is maintained in the active site by several non-polar interactions with residues of the PaAhpC active site microenvironment comprising three subunits of the decamer. Curiously C2 possess physicochemical characteristics that resemble organic peroxides found in biological systems that are derived from aromatic compounds or long chain molecules, such as nitrogenous bases and lipids hydroperoxides and, ultimately, may mimics these kinds of biological substrates. Altogether, our results reveal a novel class of inhibitory natural compounds able to exert activity over the bacterial PaAhpC.

General
Chromatographic separation procedures were performed using silica gel (Merck, 230-400 mesh) and silica gel 60 PF 254 (Merck) for column separation and for analytical (0.50mm) TLC, respectively. NMR spectra were recorded on a Varian Inova 500 spectrometer, operating at 500 MHz ( 1 H nuclei) and 125 MHz ( 13 C nuclei). CDCl 3 (Aldrich) was used as a solvent whereas tetramethylsilane (TMS) was employed as an internal standard. ESI-HRMS were recorded on a Bruker Daltonics MicroTOF QII spectrometer acquired with ESI (electrospray ionization) in both positive and negative ion mode. All solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Thermo Scientific (Logan, UT, USA).

Plant material
Branches of P. crassinervium were collected at Parque Estadual Fontes do Ipiranga, São Paulo State, Brazil and identified by Dr. Guilherme M. Antar from the Instituto de Biociências of Universidade de São Paulo (IB-USP), receiving the registration code SISGEN A4123E4. A voucher specimen (SPF 218827) was deposited in the Herbarium of IB-USP, São Paulo, SP, Brazil.

Extraction and isolation of the constituents
Dried and powdered branches (85 g) were exhaustively extracted with n-hexane at room temperature. The solvent of combined n-hexane was evaporated under reduced pressure to afford 747 mg of crude extract. Part of this material (670 mg) was chromatographed over a silica-gel column, eluted with increasing amounts of EtOAc in n-hexane, to afford pure C1 (79.5 mg) and C2 (44.2 mg).

Determination of enzyme concentrations
Protein concentrations were measured by spectrophotometry at λ = 280 nm by the molar extinction coefficient using the ProtParam tool (http://web.expasy.org/protparam). The enzyme abbreviations, Uniprot codes and enzyme properties are presented in Table 1.

Treatment of the enzymes with compounds C1 and C2
Enzyme samples [100 μM] were reduced with 5mM dithiothreitol (DTT) for 30 minutes at 25˚C and desalted using PD10 columns (Cytiva Life Sciences, Marlborough, MA, USA), quantified, and the protein concentration were adjusted to 10 μM and treated with 50 M equivalents of compounds C1 or C2 for one hour at room temperature. As control of cysteine alkylation/ enzyme inactivation, the Prx were treated with N-ethylmaleimide (NEM) 1h/RT. Finally, the treated proteins were desalted again to remove excess of the compounds and quantified again for use in subsequent assays.

NADPH coupled oxidation assay to test inhibitory properties of compounds C1 and C2
The inhibitory properties of compounds C1 and C2 on PaAhpC and SeAhpC peroxidase activity were tested using the E. coli Trx system by the coupled NADPH oxidation assay as previously described [22]. Briefly: 3 μM of PaAhpC or SeAhpC was added to a mix containing 6 μM of EcTrx, 0.9 μM of EcTrxR, 150 μM of NADPH, in 50 mM HEPES pH 7.4 buffer, 1 mM DTPA and 100 μM sodium azide, and then incubated for 5 minutes at 37˚C. The reactions were initiated by the addition of H 2 O 2 (500 μM) and NADPH decay were monitored spectrophotometrically at 340 nm. As negative controls are represented by reactions without AhpC are (negative control), or AhpC pretreated with NEM (40 μM) (inhibition control), proteins without pretreatment (positive control), and proteins pretreated either with compounds C1 and C2. To evaluate the inhibitory activity of compound C2 over the human peroxiredoxin isoform were used human HsPrx2 (5 μM) and the heterologous yeast Trx system (ScTrx1, 10 μM and ScTrxR1, 0.2 μM).

Analysis of protein sequences and crystallographic structures
Protein alignments were performed using Clustal O [36], and the graphical representations were generated using JalView [37]. Molecular crystallographic structures representations were made using the PyMol (https://pymol.org/2/).

Molecular docking approaches
Molecular dockings were performed using the coordinates 4MA9 from S. typhimurium AhpC obtained from the Protein Data Bank. Three-dimensional structures of compounds C1 and C2 were constructed using MolView [38]. Docking simulations were performed using AutoDock Vina [39,40], griding the active site region (20 × 20 × 20Å), using the deprotonates Cys P (thiolate state). Were generated 15 positions per simulation. Each ligand orientation was analyzed using UCSF Chimera tool [41] to determine the best pose using as criterion the distance of the reactive Cys P and the α,β-unsaturated group of the compounds with maximum distance of 5.0 Å and also the position compared with ligands found in the crystallographic structures of Prx isoforms active site as H 2 O 2 (PDB code = 3A2V), benzoate (2V32 1HD2; 1OC3; 2V41; 1H4O) and tert-butylbenzene-diol (4K7O). The protein-ligand analysis interactions of the best poses were performed using the LigPlot+.

Evaluation of inhibitory properties of compounds C1 and C2
To assess the inhibitory activity of compounds C1 and C2, we employed the NADPH oxidation assay [29] using the recombinant AhpC from P. aeruginosa (PaAhpC) or S. epdidermidis (SeAhpC) and the heterologous Trx system from E. coli. Representative results of the PaAhpC, SeAhpC, EcTrx, and EcTrxR expression and purification procedures are shown in S7A-S7D Fig. As inhibition control, reduced samples of PaAhpC or SeAhpC were treated with NEM, a well-known and powerful cysteine alkylating agent that is able to annihilate peroxiredoxin activity [44]. As expected for the samples subjected to NEM treatment, no peroxidase activity was detected to both enzymes as indicated by the initial rates of hydrogen peroxide decomposition when compared to the enzyme without treatment (Fig 3 and Table 2).
Regarding the tested natural products, compound C1 was not able to interfere significatively on the peroxidase activity of both AhpCs (Fig 3A and 3C; Table 2). Conversely, while the compound C2 nearly abolished PaAhpC enzyme activity, no significant effect was observed on SeAhpC (Fig 3B and 3D; Table 2). We also compared the residual amounts of NADPH after 300 seconds of reaction, and the results confirmed that compound C2 was able to inhibit the peroxidase activity of PaAhpC but not of SeAhpC (S8 Fig).
The difference in the inhibitory properties of compound C2 over the AhpCs of P. aeruginosa and S. epidermidis may rely on differences in enzyme structure. It has been shown that PaAhpC and SeAhpC present distinct properties including redox structural switches (dimer ! decamer transition) and peroxidase activity over distinct hydroperoxides which were correlated to the single amino acid substitution in the catalytic triad [22], suggesting that structural peculiarities may impact the interaction with the substrates.

Determination of IC 50 of compound C2 to PaAhpC
Since the compound C2 was able to inhibit the peroxidase activity of PaAhpC, we proceeded to determine the apparent IC 50 of the enzyme. For this, previously reduced enzymes samples were treated with different stoichiometric ratios of compound C2 (5, 10, 25, 50, and 100 molar equivalents). Excess was removed and the PaAhpC was evaluated by the NADPH oxidation assay (Fig  4A). Our results revealed that the amount of compound C2 necessary to inhibit the PaAhpC was approximately 20 μM (Fig 4B), a high value for an inhibitor but similar to the amount of the compound adenanthin (15 μM), another natural inhibitor to the human Prx2 [29].

Evaluation of specificity over the Prx human isoform and oxidoreductases thiol proteins
Since compound C2 did not exert a significant inhibitory effect on SeAhpC, we evaluated if the compound was able to inhibit other thiol proteins. Initially, we evaluated the inhibitory effect over Prx2 from humans (HsPrx2), the host of P. aeruginosa, and Trx and TrxR from bacteria, since some inhibitors described to eukaryotic Prx also inhibit the two latter thiol proteins [26, 45,46]. To perform the assays PaAhpC and SeAhpC samples were previously reduced with DTT (5 mM/1h). The DTT excess was removed, and the enzymes were treated with 40 molar equivalents of C1 (A and C, red square) or C2 (B and D, red square) for 1hour at room temperature. The compounds excess was removed, and the peroxidase activity was accessed by the NADPH oxidation in reactions containing: 3 μM of PaAhpC or SeAhpC, 6 μM EcTrx, 0.9 μM EcTrxR and 150 μM NADPH in buffer 50 mM HEPES (pH = 7.4), 100 μM DTPA and 1 mM sodium azide. Reactions were incubated at 37˚C for 5 minutes before being initiated by the addition of H 2 O 2 (500 μM) and monitored spectrophotometrically at 340 nm, 37˚C, for 5 minutes. Reactions containing PaAhpC or SeAhpC without natural compounds treatment (green square) were used as positive controls for peroxidase activity, and reactions without AhpC (black square) were used as negative controls. PaAhpC or SeAhpC samples previously treated with NEM were used as control of protein inhibition (blue square). All experiments were performed at least three times in triplicate.
https://doi.org/10.1371/journal.pone.0281322.g003 We expressed and purified the HsPrx2 and used the heterologous Trx system from S. cerevisiae (ScTrx1 and ScTrxR1) (S7E- S7G Fig) to perform the peroxidase activity assays by NADPH oxidation assay, as described previously by Truzzi and coworkers [35]. The need for the heterologous yeast system lies in the fact that human TrxR is a seleno protein able to decompose hydroperoxides [47,48], and the use of the human Trx system would make the results unreliable. The NADPH oxidation assay was performed using the same experimental conditions applied to the Prx bacterial isoforms, and the results revealed that compound C2 was not able to inhibit the human isoform HsPrx efficiently, since only a very slight decrease in peroxidase activity was detected (Fig 5A, Table 3).
Concerning the thiol proteins from E. coli, compound C2 was not able to significantly affect the activity of EcTrx or EcTrxR (Fig 5B). Only a very slight decay of Trx activity was detected and no inhibition was observed for EcTrxR. We then compared the residual amounts of NADPH after 300 seconds of reaction, and the results show that C2 was not able to affect the enzymatic activity of HsPrx2, EcTrx, and EcTrxR (S9 Fig). These results indicate there is a selectivity of compound C2 for PaAhpC when compared to other thiol proteins. This finding is important since some Prx inhibitors are able to inhibit other thiol enzymes, including different Prx isoforms and the Trx system enzymes [25,26,29,30,45,46].

Mode of PaAhpC inhibition
To better understand the mode of inhibition, we used PaAhpC pretreated with compound C2 (50 μM), the excess was removed and the enzyme was challenge with different concentrations of hydroperoxides (from 50 to 750 μM). Although the C2 concentration of 50 μM is able to strongly inhibit the peroxidase activity of PaAhpC, if the mode of inhibition is a competitive behavior, the growing concentrations of peroxides may be able to shift the compound C2 away from the active site and restore the peroxidase PaAhpC activity. Our results revealed that only a residual activity was observed (Fig 6A). Since the amount of inhibitor was not able to overwhelm the activity of all the enzyme population, the portion of the enzyme that had not reacted with the inhibitor would maintain its normal kinetics. The determination of kinetic  parameters revealed that no significant effect was observed in the K m . On the other hand, parameters as K cat and V max were expressively impacted (Fig 6B; Table 4). This behavior is compatible with an irreversible inhibitor as expected by a thiol-Michael addition chemistry [29, [49][50][51].

Investigation of Michael addition adducts formation
Some natural products identified as inhibitors of human Prx exert their biological activity via thiol-Michael addition reaction by alkylating the catalytic cysteines [29,30,33]. Compounds C1 and C2 tested in this work possess functional groups able to perform a Michael addition, i.e., an α,β-unsaturated carbonyl group. However, while compound C1 showed no significant inhibitory effect, compound C2 was able to inhibit PaAhpC, suggesting that the form of inhibition may occur by other means.
To evaluate if the inhibitory mechanism of compound C2 to PaAhpC occurs through the thiol-Michael addition, we performed non-reducing SDS-PAGE, taking advantage of the difference in gel migration of the protein in different oxidation states. When oxidized to disulfide, AhpC enzymes migrate as a dimer (~44 kDa) due to intermolecular disulfide bond formation. On the other hand, the reduced enzyme appears as a monomer (~22 kDa) [52]. Consequently, it is possible to evaluate not only the redox state of these enzymes but also if the inhibitor was able to react with cysteine residues involved in catalysis. If one of the two cysteines is covalently modified by an inhibitor, no intermolecular disulfide is expected to be formed if the enzyme is challenge with hydroperoxides, and the proteins are detected as monomers.
Prior to peroxide treatment, PaAhpC samples were reduced by DTT (Fig 7, lane 2), the excess was removed (Fig 7, lane 3), and the PaAhpC proteins were oxidized with hydrogen peroxide (Fig 7, lane 4). As expected, PaAhpC reduced by DTT were detected as monomers, while the oxidized samples were detected as dimers. As internal control of the inhibition of the disulfide formation, PaAhpC sample was previously treated with NEM and then oxidized by hydrogen peroxide.
The NEM treatment prevented disulfide formation and the proteins migrated as monomers (Fig 7, lane 5). Regarding samples pre-treated with compound C2 (Fig 7, lanes 6 and 7), the PaAhpC samples were also detected as dimers, even at very high concentration (e.g. 100 molar equivalents), revealing the formation of the intermolecular disulfide and indicating the mode of inhibition does not occur by the Michael addition mechanism over the protein thiols. This suggests that compound C2 is able to inhibit PaAhpC in a manner distinct from those already described for other Prx inhibitors from eukaryotes [29, 30, 33] and bacteria [25,26]. Although minutes. The positive controls contained the proteins HsPrx2, EcTrx or EcTrxR without prior treatment with compound C2 (green square) and the negative control without the addition of PaAhpC (EcTrx or EcTrxR) (black square). The enzymes samples previously treated with NEM were used as control of protein inhibition (blue square). The experiments were performed three times using triplicates.

Sample No enzyme (Negative control) Enzyme + NEM (Inhibition control) Enzyme (Positive control) Enzyme + C2
HsPrx2 our data suggests that the PaAhpC cysteine is not alkylated by compound C2, another reactive groups as amine (-NH 2 ) and alcohol (-OH) can perform the Michael addition chemistry (e.g. aza-and oxa-Michael addition, respectively) [53,54]. Both groups are present in the side chains of the PaAhpC catalytic triad residues Arg and Thr and are involved in substrate targeting and stabilization (Fig 1) and the modification of either group by C2 may have a strong impact in enzyme activity.

Molecular docking approaches
To assess molecular interactions between compound C2 and PaAhpC, molecular docking simulations were performed using the structure of AhpC from S. typhimurium, since AhpC from P. aureginosa (PaAhpC) has no determined crystallographic structure. The P. aeruginosa and S. typhimurium isoforms are highly related, presenting 75% of similarity and 60% identity (Fig 8).
Docking approaches were performed using the decameric structure once the decamer is in the active form, whose geometry is optimal for the productive binding of the peroxide substrates. The AhpC active site environment is composed of residues of three protomers: the obligate dimer and the monomer of the neighboring dimer (Fig 9A).
The solutions were distributed mostly in the microenvironment of the active site in close proximity to the residues involved in catalysis (Fig 9B). The best pose was selected based on the free energy of Gibbs (ΔG = -7.2 kcal/mol) and superposition with ligands found in the active site of Prx crystallographic structures, such as the oxidant substrate (hydrogen peroxide in Aeropyrum pernix Prx) [55] and compounds with structural similarities to C2 compound  (benzoic acid found in the Arenicola marina Prx6 and Homo sapiens Prx5 and tert-butylbenzene-diol present H. sapiens Prx5) [56][57][58] (Fig 9C). quotation (") symbols denotes residues form the obligate dimer, and the asterisk ( � ) denotes the adjacent protomer dimer) (Fig 9D).

Stabilization of compound C2 mostly occurred by hydrophobic interactions and van der
The functional importance of some residues identified in the docking procedures has been determined. It has already been shown that the Phe/Tyr residue at this position in peroxiredoxins is involved in the decamer stabilization of the enzyme, through an unconventional hydrogen bond with the catalytic triad Thr [22,23,59,60]. Additionally, Glu 138 would be related to conformational changes during the catalytic cycle [61]. Except for the Val 45 and Glu 138 of StAhpC that are substituted by Asn in PaAhpC, a residue with similar physicochemical properties, all the remaining interacting residues are strictly conserved among the two peroxiredoxins, which corroborates the feasibility of the docking analysis.
An important aspect about the mode of inhibition, which was irreversible, may have been related to the adoption of Michael with other groups such as the O and N atoms from the side chain of the catalytic triad Thr of Arg residues. The α,β-unsaturated carbonylic system is in close proximity to both reactive group residues of these amino acids, which are also more exposed than Cys P and involved in interactions to guide the substrates to catalytic cysteines (Fig 1). Nonetheless, these assumptions lack experimental support and the determination of PaAhpC structures with compound C2 is needed to reach any meaningful conclusions.

Discussion
We tested two biosynthetic related natural products isolated from P. crassinervium as inhibitor of the AhpC from bacteria: 4-hydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzoic acid (C1), and 4-hydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzoic acid (C2). Both compounds possess three common structural aspects: 1) benzene rings, 2) an α,β unsaturated carbonylic system, and 3) a flexible tail (oxo-geranyl unity) that resembles long-chain fatty acids. The sole difference is a hydroxyl group at C-4 position in compound C1 which is substituted by a carboxylic acid in compound C2 (Fig 2). Therefore, it is reasonable to assume that the carboxyl group is involved in the inhibitory properties of compound C2. In fact, benzoic acids and derivatives have been found in some crystallized in different peroxiredoxin structures, suggesting that the carboxylic acid in the aromatic ring is a factor that may favor the stabilization of molecules in the active site [56,57,62].
The hydrophobic nature, including the presence of one aromatic ring, places compounds C1 and C2 with other chemically related molecules identified for this group of proteins [29,30,32,33,63,64]. Although the oxo-geranyl unity has not been observed in previously described inhibitors, it resembles substrates such as long-chain fatty acids hydroperoxides (oleic and linoleic acid peroxides, for example) which are believed to be biological substrates of some thiol peroxidases including Prx [65,66]. The docking approaches presented here reveal that the tail may be stabilized in the active site pocket by several hydrophobic interactions with non-polar residues. This feature appears to be important and can be exploited in the search for natural inhibitors for Prx. Unlike other inhibitors that also present an α,β-unsaturated carbonyl system, neither compound C1 and C2 perform a thiol-Michael addition reaction over the reactive cysteines, revealing a mode of inhibition distinct from those previously characterized with other natural compounds to the human isoforms [29,30]. This is an important aspect since most of the Prx inhibitors described to date perform an inhibition by means of chemical reactions involving a covalent bond between ligand and the thiol of the cysteines but with low specificity, affecting other thiol proteins and being very toxic to the cells [25, 45,46]. Conversely, the compound C2 shows high specificity over PaAhpC when compared to SeAhpC, HsPrx, or even other thiol proteins as the Trx system which are strongly inhibited by other Prx inhibitors [26, 45,46]. The specificity over PaAhpC is a very positive finding, since this enzyme is a P. aeruginosa virulence factor, and this pathogenic bacterium is related with nosocomial infections and high mortality rates [67,68]. In summary, our results demonstrate the inhibitory activity of a new class of natural products that possesses distinct characteristics from other compounds identified as peroxiredoxin inhibitors. This contribution on molecules able to exert inhibitory activity on Prx of bacteria can allow the selection of natural products or even lead to the synthesis of inhibitors, aiming to increase the specificity and inhibitory activity over the Prx in order to combat infectious and genetic diseases.